Laser scanning cytometry (“LSC”) is a technology where one or more laser beams are scanned across an analysis surface which typically contains cells or tissue. Photomultiplier tubes and photodiodes are used to detect fluorescent light emitted from the samples as well as modifications to the interrogating laser light. The outputs of the detectors are digitized, and synchronous movements of a computer controlled microscope stage allow accumulation of computer memory arrays of detector outputs that can be treated as images of the areas of the specimen scanned. The memory arrays differ from camera-based images in that there is not a one-to-one correspondence between the pixel areas of the image and the physical area of the slide; instead, a variable-sized evaluation area is centered about the pixel location. The array “images” are segmented by a number of methods to identify events of interest. Quantitative data is calculated for each event and multi-feature data is analyzed for each of many thousands of events in a typical analysis.
U.S. Pat. Nos. 5,072,382 and 5,107,422 describe the general operation of laser scanning cytometers. U.S. Pat. No. 6,002,788 describes details of laser light scatter, light loss and absorbance measurements. Each of the above patents are hereby incorporated here by reference.
Light scatter and absorption may also be measured by a LSC system using a photodiode detector. In accordance with one such system, a blocker bar is placed between a laser beam and a detector. When a cell or other object interferes with the laser beam, light scattered by the object bypasses the blocker bar and strikes the detector, producing an increased signal. The resultant image has a dark background with bright areas where cells or other objects are present. This type of light scatter is analogous to the light scatter used in flow cytometry and is often used for the initial identification of cells.
A variation of light scatter measurement may be used to obtain bright field images of cells with a high degree of morphological detail. This is accomplished by varying the position of the blocker bar to allow a portion of the laser beam to impinge on the detector at all times. The signal produced by the portion of the laser which impinges on the detector at all times serves as a reference signal. As cells and other objects interact with the laser beam, structures within them scatter and/or absorb light and modulate the strength of the reference signal. (An example of such an LSC and system is described in U.S. Pat. No. 6,002,788.)
Another variation of laser light measurement is the “light loss mode.” In accordance with this variation, no blocker bar is employed. The laser beam continuously impinges on the detector and produces a high reference signal. When objects interact with the beam signal strength is diminished. Refractile objects, such as beads and spherical cells, will refract light away from the detector and chromatically stained objects, such as cells in a tissue section, will absorb the laser light. In both cases bright-field images are produced with dark objects. These images are often digitally inverted so that they can be analyzed in a manner similar to fluorescence-based analysis. (An example of such an LSC and system is described in co-pending U.S. patent application Ser. No. 11/040,183, entitled “Method and Device for Interrogating Samples Using Laser Scanning Cytometry and Other Techniques” and filed Jan. 21, 2005, which is hereby incorporated herein by reference.)
Most laser scanning cytometers are equipped with multiple lasers to excite a wide variety of fluorescent dyes. Often this analysis is done in a multiplexed fashion, where a scan area is first scanned with one color laser and then the same scan area is scanned with a second color laser. The data from both scans are combined and images are interchangeable. (An example of a LSC system employing multiple lasers is described in U.S. Pat. No. 5,885,840, which is hereby incorporated herein by reference.)
In accordance with multiple laser LSC systems, for each scan pass, laser scatter or absorption can be obtained. Chromatic dyes absorb light at different portions of the electromagnetic spectrum, with the combination of the interrogating wavelengths and the dyes' absorption spectral response giving the dyes their distinctive colors. For each laser used, there will be differential absorption of the beam by the different dyes used to the stain the sample. In a standard iCyte® LSC system (manufactured by Compucyte Corporation of Cambridge, Mass.), blue laser absorption can be obtained along with red laser absorption, as seen in FIGS. 1A and 1B.
As noted above, multi-color fluorescence technology has developed, largely in the area of flow cytometric analysis. Research-grade instruments are capable of measuring up to 12 colors of fluorescence on individual cells using a combination of multiple excitation lasers and a plurality of photomultiplier tubes coupled to discrete bandwidth filters. One problem encountered in performing multi-color fluorescence analysis is spectral overlap, where the fluorescence emission spectrum of a dye extends into the bandwidths measured by several detectors. Compensation techniques have been developed that can correct for this spectral overlap by taking a proportion of the signal from an interfering dye's detector and subtracting it from the signal being quantified.
In the biological arts, tissue analysis is often performed using sections of tissues that have been stained with chromatic dyes. Such techniques are often applied in connection with research pathology, drug discovery and validation, biomarker discovery, and drug safety procedures based on tissue analysis. Chromatic dyes are traditionally examined by techniques related to bright field microscopy, and methods of evaluating chromatically stained samples include (1) manual scoring (0, to +++), depending on various factors including the staining intensity and the number of cells stained and (2) automated image analysis techniques using images obtained by digital photo-microscopy of samples where the optical density measurements are used as the metric.
One of the inherent problems in undertaking quantitative analysis of tissue sections is the fact that tissues are heterogeneous in nature, and they often contain varying levels of either endogenous or preparation-associated auto-fluorescence. This auto-fluorescence is known to interfere with fluorescence analysis. Correction for auto-fluorescence is a distinct process, different from spectral overlap correction. Methods to correct for the interference of auto-fluorescence associated with fluorescence using multiple wavelength laser excitation are known in the art. (See, for example, Lee, M., Luther, E. (2004). “Using virtual channels to perform compensation and correct background autofluorescence in laser scanning cytometry.” ISAC XXII International Congress. Cytometry Part A 59A(1): 27-73.
Methods have also been described to convert color camera RGB or HSL values to dye equivalents. See, for example, U.S. Pat. No. 6,819,787 issued to Stone et al. and Ruifrok et. al., Comparison of Quantification of Histochemical Staining by Hue-Saturation-Intensity (HIS) Transformation and Color-Deconvolution. Applied Immunohistochemistry and Molecular Morphology, vol. 11(1), pp. 85-91, March 2003. However, these methods have the disadvantage that broad spectrum light is used as the light source, resulting in less control of the spectral characteristics of the fluorochromes being evaluated.